Bacterial contamination of blood products continues to be the major microbiological cause of transfusion-associated morbidity and mortality. Platelets are the most susceptible blood product to bacterial contaminants. Furthermore, platelets are generally stored aerobically for up to 5 days at 20 to 24° C., allowing a wide variety of bacteria to grow.
Conventional methods for the detection of bacteria in blood components involve culturing and identification by morphological, biochemical, and immunological characteristics. Several methods have been designed to detect bacterial contamination in platelets with different ranges of sensitivity and specificity. The automated culture system BacT/ALERT™ has been implemented for routine testing of bacterial contamination of platelets in Canada and by many blood suppliers from the United States and Europe. Although BacT/ALERT™ can detect <10 colony forming units (cfu)/ml of bacteria, this system detects microorganism growth by tracking CO2 production via a calorimetric sensor-and-detection system, and thus provides non-specific detection of bacterial presence. Microorganisms multiply in a select media, generating CO2 and as CO2 increases, a calorimetric sensor is indicated, typically in a bottle. Measuring reflected light, the system monitors and detects color changes in the sensor, and algorithms are used to analyze the data to determine positivity.
According to this system, changes in the sensor are permanent and visible to the unaided eye. Despite its high sensitivity, this system lacks specificity, requires a high volume of platelets for testing, and involves long incubation periods for bacterial detection.
Likewise, a commercially available kit for nucleic acid detection of microbial contaminants known as Bug's n' Beads™ is described in International Publication WO 98/51693 published Nov. 19, 1998. Although Bugs n' Beads™ provides a different approach to nucleic acid detection, it does so in a time consuming manner, while requiring a generous sample size.
Other molecular genetic techniques have been employed for detecting bacterial contamination at sensitive levels for some bacterial species. Dreier et al. (Journal of Clinical Microbiology, 2004, Vol. 42, p. 4759-4764) describe a Real-Time Reverse Transcriptase Polymerase Chain Reaction (PCR) Assay for detecting bacterial contamination in platelets. This methodology targeted a 290-bp product of the 23S rRNA as a broad target in the detection of a diversity of bacterial species. However, the sensitivity of this methodology was marginal, even with the employment of 7 ml of platelet-rich plasma as a starting material. As a result, further optimization achieving a larger nucleic acid input was required.
Since the maximum storage period for platelets is 5 days, and bacterial contamination of platelets is of growing concern in donor blood products, there remains an eminent need for a rapid, sensitive and efficient method for detecting and quantifying bacterial contamination in platelet samples.